【作者】 张华；

【导师】 俞汝勤；

【作者基本信息】 湖南大学 ， 分析化学， 2013， 硕士

【摘要】 酶是活细胞内产生的具有高度专一性和催化效率的蛋白质，生物体在新陈代谢中，几乎所有的化学反应都是在酶的催化下进行的，酶是生命活动必不可少的物质，人体一旦缺失酶会导致多种遗传疾病。因此，酶活性的测定在临床医学上具有十分重要的现实意义。近年来，生物传感器以其特异性强、分析速度快、操作简单、成本低等优点而广泛应用于生物医学、食品、化学、环境监测和医药等领域。因此，本论文以腺苷脱氨酶、尿嘧啶DNA糖基化酶和T4多聚核苷激酶为检测对象，发展了一系列新型的生物传感技术。具体内容包括：腺苷脱氨酶（ADA）是一种重要的核酸代谢酶，其表达含量是临床上一些重大疾病的诊断指标之一。第2章中，我们首次开发了一种基于酶调控非标记纳米金团聚的策略用于ADA的比色检测。该方法主要依赖于腺苷的环外氨基与纳米金形成强烈的相互作用，从而将纳米金表面的柠檬酸根离子置换下来，导致纳米金团聚，纳米金溶液颜色由红色变为紫色。而ADA对腺苷进行脱氨基作用后，生成的次黄苷不能与纳米金表面形成相互作用，从而不会引起纳米金团聚，纳米金溶液仍为红色。因此，通过纳米金溶液颜色的变化即可实现对ADA的可视化检测。该方法操作简单、响应快速，并且具有较高的灵敏度，其检测下限为0.8227U/L。此外，我们利用该方法成功实现了对其抑制剂的测定。作为一种重要的碱基切除修复酶，尿嘧啶DNA糖基化酶（UDG）在DNA损伤修复上发挥着重要作用。第3章中，我们开发了一种结合酶放大的电化学传感平台用于UDG活性及其抑制剂的简单灵敏检测。该测定方法依赖于UDG作用后双链UDG底物的熔链温度降低，从而导致生物素化的信号探针从电极表面脱离。信号探针上标记的生物素用作示踪物，碱性磷酸酯酶标记的链霉亲和素（SA-ALP）用作报告分子。标记生物素的信号探针的失去导致电极表面结合的SA-ALP数量减少，因此造成产生的电信号减小。该测定方法简单、成本低廉、选择性好，具有较宽的响应范围（0.01-10U/mL）和较低的检测下限（0.0079U/mL）。该方法不仅提供了一种通用的碱基切除修复酶的测定方法，而且还在药物筛选上显示出了潜在的应用价值。T4多聚核苷激酶（T4PNK）对DNA的5′羟基磷酸化修饰在DNA断裂修复上发挥着重要作用。第4章中，我们建立了一种目标激活自催化DNA酶放大的策略用于T4PNK的测定。首先我们用T4PNK将一段E6DNA酶的互补序列的5′羟基末端磷酸化，磷酸化后的序列与E6DNA酶杂交使DNA酶活性被抑制，加入λ核酸外切酶（λexo）后，5′磷酸化的互补序列被降解，E6DNA酶游离出来激活自催化信号放大过程。而当不存在T4PNK时，互补序列不能被磷酸化，从而导致后续λexo降解和DNA酶的自催化放大过程都无法进行。该方法利用E6DNA酶的自催化信号放大过程，实现了对T4PNK的灵敏检测。更多还原

【Abstract】 Enzymes are a kind of proteins produced in living cells that have high specificity and catalytic efficiency. Enzymes are responsible for accelerating almost all chemical reactions of metabolism in organism. They are the indispensible materials to sustain life, and enzyme deficiency is highly related to many hereditary diseases. Therefore, the detection of enzyme activity has very important realistic meanings in clinical medicine. In recent years, biosensors have been widely applied in biomedical, food, chemical, environmental monitoring and medical fields, attributing to the advantages of high specificity, rapid response, easy operations and cost-effectiveness. This thesis developed a series of novel biosensing technology for the detection of adenosine deaminase (ADA), Uracil-DNA glycosylase (UDG) and T4polynucleotide kinase (T4PNK). The details are described in following chapters.Adenosine deaminase (ADA) is one of important DNA metabolic enzyme, its expression is one of the important indexes for diagnosis of several critical diseases in clinic. In Chapter2, we first developed a colorimetric strategy for the detection of ADA based on enzyme-mediated label free gold nanoparticle (GNP) aggregation. This assay relies on the fact that the exocyclic amino of adenosine can strongly interact with GNP, which would displace the citrate from the GNP surface, and result in the aggregation of GNP with the solution color changes from red to purple. When adenosine was treated with ADA, the resultant inosine would not interact with the GNP surface as well as the GNP aggregation, as a result the solution was still red. Visual detection of ADA could be realized by monitoring the color changes of the solution. The proposed method is easy operation, rapid response and high sensitivity, the detection limit is down to0.8227U/L. In addition, the proposed strategy was also applied to the inhibitor assay.As an important base excision repair (BER) enzyme, uracil-DNA glycosylase (UDG) plays a crucial role in DNA lesion repair. In Chapter3, an electrochemical sensing platform combined with enzymatic amplification was developed for simple and sensitive assay of UDG activity and its inhibition. This strategy relies on the release of a biotinylated signal probe from the electrode surface, due to the lowered melting temperature of the duplex UDG substrate after UDG treatment. A biotin modification was used as a tracer in the signal probe and streptavidin-alkaline phosphatase (SA-ALP) was taken as a reporter molecule. The loss of biotin label led to a decrease in the amount of bound SA-ALP on the electrode surface, resulting in a weaker electrochemical signal. This strategy allowed for a simple, cost-effective, sensitive and selective assay for UDG with a wide linear response range from0.01to10U/mL and a low detection limit of0.0079U/mL. The proposed strategy not only provides a universal platform for the assay of BER enzymes, but also shows potential application for drug screening.The5’-hydroxyl phosphorylation of T4polynucleotide kinase (T4PNK) plays an important role in DNA break repair. In chapter4, we developed a novel target-triggered self-catalytic DNAzyme amplification method for the detection of T4PNK. The5’-hydroxyl end of the complementary sequence of E6DNAzyme was firstly phosphorylated using T4PNK, and then the phosphorylated sequence hybridized with E6DNAzyme in order to inhibit its activity. Upon treatment with lambda exonuclease (λexo),5’-phosphorylated sequence was digested and release the E6DNAzyme, leads to the activation of self-catalytic DNAzyme amplification. In the absence of T4PNK, the complementary sequence of E6DNAzyme would not be phosphorylated, and then the digestion ofλexo as well as the self-catalytic amplification of DNAzyme would not occur. By taking advantage of the self-catalytic DNAzyme amplification, the sensitive detection of T4PNK could be realized.更多还原